This invention relates to nucleic acid vaccines for rickettsial diseases of animals, including humans.
The rickettsias are a group of small bacteria commonly transmitted by arthropod vectors to man and animals, in which they may cause serious disease. The pathogens causing human rickettsial diseases include the agent of epidemic typhus, Rickettsia prowazekii, which has resulted in the deaths of millions of people during wartime and natural disasters. The causative agents of spotted fever, e.g., Rickettsia rickettsii and Rickettsia conorii, are also included within this group. Recently, new types of human rickettsial disease caused by members of the tribe Ehrlichiae have been described. Ehrlichiae infect leukocytes and endothelial cells of many different mammalian species, some of them causing serious human and veterinary diseases. Over 400 cases of human ehrlichiosis, including some fatalities, caused by Ehrlichia chaffeensis have now been reported. Clinical signs of human ehrlichiosis are similar to those of Rocky Mountain spotted fever, including fever, nausea, vomiting, headache, and rash.
Heartwater is another infectious disease caused by a rickettsial pathogen, namely Cowdria ruminantium, and is transmitted by ticks of the genus Amblyomma. The disease occurs throughout most of Africa and has an estimated endemic area of about 5 million square miles. In endemic areas, heartwater is a latent infection in indigenous breeds of cattle that have been subjected to centuries of natural selection. The problems occur where the disease contacts susceptible or naive cattle and other ruminants. Heartwater has been confirmed to be on the island of Guadeloupe in the Caribbean and is spreading through the Caribbean Islands. The tick vectors responsible for spreading this disease are already present on the American mainland and threaten the livestock industry in North and South America.
In acute cases of heartwater, animals exhibit a sudden rise in temperature, signs of anorexia, cessation of rumination, and nervous symptoms including staggering, muscle twitching, and convulsions. Death usually occurs during these convulsions. Peracute cases of the disease occur where the animal collapses and dies in convulsions having shown no preliminary symptoms. Mortality is high in susceptible animals. Angora sheep infected with the disease have a 90% mortality rate while susceptible cattle strains have up to a 60% mortality rate.
If detected early, tetracycline or chloramphenicol treatment are effective against rickettsial infections, but symptoms are similar to numerous other infections and there are no satisfactory diagnostic tests (Helmick, C., K. Bernard, L. D""Angelo [1984] J. Infect. Dis. 150:480).
Animals which have recovered from heartwater are resistant to further homologous, and in some cases heterologous, strain challenge. It has similarly been found that persons recovering from a rickettsial infection may develop a solid and lasting immunity. Individuals recovered from natural infections are often immune to multiple isolates and even species. For example, guinea pigs immunized with a recombinant R. conorii protein were partially protected even against R. rickettsii (Vishwanath, S., G. McDonald, N. Watkins [1990] Infect. Immun. 58:646). It is known that there is structural variation in rickettsial antigens between different geographical isolates. Thus, a functional recombinant vaccine against multiple isolates would need to contain multiple epitopes, e.g., protective T and B cell epitopes, shared between isolates. It is believed that serum antibodies do not play a significant role in the mechanism of immunity against rickettsia (Uilenberg, G. [1983] Advances in Vet. Sci. and Comp. Med. 27:427-480; Du Plessis, Plessis, J. L. [1970] Onderstepoort J. Vet. Res. 37(3):147-150).
Vaccines based on inactivated or attenuated rickettsiae have been developed against certain rickettsial diseases, for example against R. prowazekii and R. rickettsii. However, these vaccines have major problems or disadvantages, including undesirable toxic reactions, difficulty in standardization, and expense (Woodward, T. [1981] xe2x80x9cRickettsial diseases: certain unsettled problems in their historical perspective,xe2x80x9d In Rickettsia and Rickettsial Diseases, W. Burgdorfer and R. Anacker, eds., Academic Press, New York, pp. 17-40).
A vaccine currently used in the control of heartwater is composed of live infected sheep blood. This vaccine also has several disadvantages. First, expertise is required for the intravenous inoculation techniques required to administer this vaccine. Second, vaccinated animals may experience shock and so require daily monitoring for a period after vaccination. There is a possibility of death due to shock throughout this monitoring period, and the drugs needed to treat any shock induced by vaccination are costly. Third, blood-borne parasites may be present in the blood vaccine and be transmitted to the vaccinates. Finally, the blood vaccine requires a cold chain to preserve the vaccine.
Clearly, a safer, more effective vaccine that is easily administered would be particularly advantageous. For these reasons, and with the advent of new methods in biotechnology, investigators have concentrated recently on the development of new types of vaccines, including recombinant vaccines. However, recombinant vaccine antigens must be carefully selected and presented to the immune system such that shared epitopes are recognized. These factors have contributed to the search for effective vaccines.
A protective vaccine against rickettsiae that elicits a complete immune response can be advantageous. A few antigens which potentially can be useful as vaccines have now been identified and sequenced for various pathogenic rickettsia. The genes encoding the antigens and that can be employed to recombinantly produce those antigen have also been identified and sequenced. Certain protective antigens identified for R. rickettsi, R. conorii, and R. prowazekii (e.g., rOmpA and rOmpB) are large ( greater than 100 kDa), dependent on retention of native conformation for protective efficacy, but are often degraded when produced in recombinant systems. This presents technical and quality-control problems if purified recombinant proteins are to be included in a vaccine. The mode of presentation of a recombinant antigen to the immune system can also be an important factor in the immune response.
Nucleic acid vaccination has been shown to induce protective immune responses in non-viral systems and in diverse animal species (Special Conference Issue, WHO meeting on nucleic acid vaccines [1994] Vaccine 12:1491). Nucleic acid vaccination has induced cytotoxic lymphocyte (CTL), T-helper 1, and antibody responses, and has been shown to be protective against disease (Ulmer, J., J. Donelly, S. Parker et al. [1993] Science 259:1745). For example, direct intramuscular injection of mice with DNA encoding the influenza nucleoprotein caused the production of high titer antibodies, nucleoprotein-specific CTLs, and protection against viral challenge. Immunization of mice with plasmid DNA encoding the Plasmodium yoelii circumsporozoite protein induced high antibody titers against malaria sporozoites and CTLs, and protection against challenge infection (Sedegah, M., R. Hedstrom, P. Hobart, S. Hoffman [1994] Proc. Natl. Acad. Sci. USA 91:9866). Cattle immunized with plasmids encoding bovine herpesvirus 1 (BHV-1) glycoprotein IV developed neutralizing antibody and were partially protected (Cox, G., T. Zamb, L. Babiuk [1993] J. Virol. 67:5664). However, it has been a question in the field of immunization whether the recently discovered technology of nucleic acid vaccines can provide improved protection against an antigenic drift variant. Moreover, it has not heretofore been recognized or suggested that nucleic acid vaccines may be successful to protect against rickettsial disease or that a major surface protein conserved in rickettsia was protective against disease.
Disclosed and claimed here are novel vaccines for conferring immunity to rickettsia infection, including Cowdria ruminantium causing heartwater. Also disclosed are novel nucleic acid compositions and methods of using those compositions, including to confer immunity in a susceptible host. Also disclosed are novel materials and methods for diagnosing infections by Ehrlichia humans or animals.
One aspect of the subject invention concerns a nucleic acid, e.g., DNA or mRNA, vaccine containing the major antigenic protein 1 gene (MAP1) or the major antigenic protein 2 gene (MAP2) of rickettsial pathogens. In one embodiment, the nucleic acid vaccines can be driven by the human cytomegalovirus (HCMV) enhancer-promoter. In studies immunizing mice by intramuscular injection of a DNA vaccine composition according to the subject invention, immunized mice seroconverted and reacted with MAP1 in antigen blots. Splenocytes from immunized mice, but not from control mice immunized with vector only, proliferated in response to recombinant MAP1 and rickettsial antigens in in vitro lymphocyte proliferation tests. In experiments testing different DNA vaccine dose regimens, increased survival rates as compared to controls were observed on challenge with rickettsia. Accordingly, the subject invention concerns the discovery that DNA vaccines can induce protective immunity against rickettsial disease or death resulting therefrom.
The subject invention further concerns the genes designated Cowdria ruminantium map 2, Cowdria ruminantium 1hworf3, Cowdria ruminantium 4hworf1, Cowdria ruminantium 18hworf1, and Cowdria ruminantium 3gdorf3 and the use of these genes in diagnostic and therapeutic applications. The subject invention further concerns the proteins encoded by the exemplified genes, antibodies to these proteins, and the use of such antibodies and proteins in diagnostic and therapeutic applications.
In one embodiment of the subject invention, the polynucleotide vaccines are administered in conjunction with an antigen. In a preferred embodiment, the antigen is the polypeptide which is encoded by the polynucleotide administered as the polynucleotide vaccine. As a particularly preferred embodiment, the antigen is administered as a booster subsequent to the initial administration of the polynucleotide vaccine.